We propose a new hypothesis to explain the regulation of solute and water exchange in individually perfused mammalian microvessels by shear stress applied to the endothelial cell glycocalyx and at the endothelial cell membrane. The overall hypothesis is that the three-dimensional ordered properties of the endothelial glycocalyx which enable it to function as the primary molecular sieve for plasma proteins also determine its function as a shear stress sensor to regulate endothelial barrier permeability. Aim 1 is to measure both small solute and large solute permeability coefficients in individually perfused microvessels when applied shear stress is varied, and the structure of the glycocalyx is maintained. Transvascular water flux and effective osmotic pressures of plasma proteins will be measured. The specific hypothesis is that an intact glycocalyx acting as a mechanotransducer and the principal molecular sieve will increase small solute permeability with no change in the permeability and selectivity to large molecules. In Aim 2 the structure of the glycocalyx is modified to reduce its action as a molecular sieve and to allow shear stress to be applied closer to the endothelial surface. The specific hypothesis is that the intact glycocalyx normally protects shear-sensing mechanotransducers at the endothelial cell membrane that regulate a common pathway for water and all solutes. Aim 3 is to perfuse microvessels for long periods at very low shear with and without an intact glycocalyx. The specific hypothesis is that continuous shear stress is required to maintain an endothelial phenotype in which an acute change in shear stress causes rapid regulation of small solute permeability, with no change in water and large solute permeability. Experimental design and interpretation will be guided by our current model of water and solute transport through the glycocalyx and inter-endothelial cleft and by a new model of force transduction from the glycocalyx to the cortical cytoskeleton. The combined microperfusion, biophysical, ultrastructural, and modeling approaches will provide new understanding of the permeability regulation by shear stress in normal microvessels and may lead to strategies to improve nutrient delivery and enhance tissue recovery after injury or surgery. Our experiments evaluate the common mechanical properties of the endothelial glycocalyx that modulate water, solute, leukocyte, and red cell fluxes at the endothelial cell surface.